Journal of Dermatological Science (2003) 33, 184 /186
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LETTER TO THE EDITOR Differential diagnosis between mycosis fungoides and chronic dermatitis by fractal analysis
The diagnosis of mycosis fungoides (MF) early stage needs a multidisciplinary approach based on clinical, histological, immunohistochemical, ultrastructural, molecular biological and genetic data [1]. Despite this, diagnostic specificity and sensitivity are not very high, and this may cause delay in the treatment of patients. Lymphoid cell nuclear contour measurement, to date evaluated by Nuclear Contour Index [NCI /perimeter/sqr(area); in the circle NCI /3.54] through ultrastructural morphometry, is among diagnostic parameters of MF; in MF NCI is assumed to be ]/7 [2]. In this study we compared the diagnostic power of NCI to that of fractal geometry applied, at transmission electron microscopy level, as a tool to analyze the nuclear contour of lymphoid cells. Fractal geometry describes the irregular and complex shape of many objects, and has successfully been used since one decade in many fields of pathology (e.g. leukemia, colorectal polyps, osteoarthrosis, etc.). We analyzed 21 IA stage MF (12 female; nine male; median age 65, range 45/78 years) and 21 cases of chronic dermatitis (CD; ten female; 11 male; median age 62, range 48/72 years) including chronic eczema (16 cases) and lichenoid reactions (five cases). Skin biopsies of erythematous patches of MF patients revealed a linear subepidermal infiltrate of CD4/ T lymphocytes, some of which showing larger hyperchromatic nuclei and epidermotropism; few intraepidermal collection of atypical lymphocytes (Pautrier’s microabscesses), as well as some eosinophil granulocytes in the infiltrate, whereas there was no relevant epidermal spongiosis. TCRg analysis documented a monoclonal T cell population in 14 MF cases, i.e. in 66.7% of 21 cases and in 2 CD, i.e. in 9.5% out of 21 cases. The ultrastructural study was carried out routinely (glutaraldehyde and osmium fixed samples, dehydration in graded alcohol, epoxy resin embedding). Ultrathin sections were examined at /5500 and images were grabbed by a b/w CCD camera and stored on floppy disks. The nuclear contour of approximately 30
randomly selected lymphoid cell nuclei was analyzed both with NCI and fractal geometry parameters. Briefly, for fractal dimension (D) analysis, the nuclear contour was extracted, resized to a standard dimension and converted to a single pixel outline (Image-Pro Plus sw., Media Cybernetics Inc., Silver Spring, USA). One pixel resulted equal to 12 nm. By the box-counting method, the fractal dimension was automatically calculated using a software written by us as follows: each image was covered by nets of square boxes (from 1 to 160 pixels) and the number of boxes containing any part of the outline was counted. Log/log graph was plotted (the reciprocal of the square side length against the number of outline containing squares), the slope of the linear fit representing the fractal dimension of the image: the more tortuous and complex was the nuclear outline, the larger was D (Fig. 1). The measuring procedure was calibrated against shapes of known fractal dimension with an accuracy of 9/1.5%. A total of 1225 images were analyzed. Differences among groups were evaluated by one-way ANOVA and linear regression analysis was used to test correlation between NCI and D variables. The resulting mean D was always greater than the topological dimension (P B/0.001) and the coefficient of determination of the linear fits had R2 value /0.95, showing the fractal approach appropriated (the mean ranges of scales for which linear log /log plot were found were between 100 and 700 nm). Both D and NCI were found to be significantly higher in MF (D /1.219/0.03; median /1.22; range 1.16 /1.28 NCI /7.69/0.6; median /7.4; range 6.7 /8.2) than in CD (D /1.119/ 0.02: median /1.11; range 1.05 /1.14; NCI / 6.19/0.4; median /5.8; range 5.6 /7.3) (Fig. 2, P B/0.001). D significantly differed between nuclei showing NCI 5/7 (D /1.099/0.02, # /705) and nuclei with NCI /7 (D /1.199/0.02; # /520) (P /0.001). A significant positive correlation was found between D and NCI (r /0.75, P B/0.001). The variance of D values was three times lower than that of NCI values (MF: 2.5 vs. 8%; CD: 1.9 vs. 6.9%, P B/0.01, P B/0.01, respectively). Fractal dimension appears to be useful for measuring the exaggerated nuclear convolutions of lymphocytes in early stage MF, and it has some
0923-1811/03/$30.00 – 2003 Elsevier Ireland Ltd. on behalf of Japanese Society for Investigative Dermatology. All rights reserved. doi:10.1016/j.jdermsci.2003.07.001
Letter to the Editor
advantages when compared with NCI. NCI is obtained from measurement of integer Euclidean values that fail to describe the complexity of the
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ultramicroscopical features, and, furthermore, its values are not comparable when performed at different magnification. Fractal geometry can be
Fig. 1 Top: Lymphoid nucleus in early MF (right) and CD (left). Transmission electron microscopy, /20 000. Middle: Nuclear outlines are covered by nets of square boxes (ranging from 1 to 160 pixels) to perform fractal analysis according to the box-counting method. Right: A complex convoluted lymphoid nucleus outline (D /1.18, MF); left: a more regular lymphoid nucleus outline (D /1.12, CD). Bottom: Log /log graphs used to calculate the box-counting fractal dimension. It can be seen that there is a central linear segment (r /0.99) and the gradient of this is taken to be the box-counting fractal dimension.
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Letter to the Editor
capability of D to discriminate between cells showing NCI values lower or higher than the MF threshold value (NCI /7); (d) the variance in each class of D data was three times lower than the variance of NCI, suggesting that the fractal approach could be more powerful than the Euclidean (NCI) approach in the diagnosis of early stage MF. We suggest that fractal analysis could be an additional tool in the challenging differential diagnosis between benign dermatitis and early stage MF.
References
Fig. 2 Box plot of fractal dimension values of lymphoid cell nuclei in CD and early MF.
a more reliable method of quantitation, independent of the magnification [3]. Although the nuclear outline, with its lower and upper limits, cannot follow a theoretical fractal, fractal analysis appears as a useful tool for distinguishing between diagnostic categories. Furthermore the analysis is neither expensive or time-consuming (1 min to measure 1 nucleus, using a 400 MHz PC Pentium). Summarizing: (a) the irregularity of lymphoid cell nuclear contour may be described by using D: this is supported by the fact that, in our study, D value was always greater than 1, and log/log plot resulted to be always as a straight-line; (b) D of the nuclear outline is significantly higher in malignant than in benign lymphoid infiltrate; (c) the measurement of 30 lymphoid cell nuclei is sufficient for reliable results, as documented by the significant correlation between D and NCI, as well as by the
[1] Parry EJ, Stevens SR, Gilliam AC, Horvath N, el-Charif M, Spiro TP, Adler LP, Shina D, Kinsella T, Heyman EN, Cooper KD, Wood GS. Management of cutaneous lymphomas using a multidisciplinary approach. Arch Dermatol 1999;135:907 / 11. [2] McNutt NS, Crain WR. Quantitative electron microscopic comparison of lymphocyte nuclear contours in mycosis fungoides and in benign infiltrates in skin. Cancer 1981;47:698 /709. [3] Losa GA, Nonnenmacher TF. Self-similarity and fractal irregularity in pathologic tissues. Mod Pathol 1996;9:174 / 82.
G. Bianciardi, C. Miracco, M.M. De Santi, P. Luzi Sez. Anatomia Patologica, Department of Human Pathology and Oncology, University of Siena, Via delle Scotte 6, 53100 Siena, Italy E-mail address:
[email protected]
